Field of the Invention
This invention relates generally to the field of immunology and, more particularly, to methods and compositions for the modulation of host immune response to an immunogen, particularly exogenous antigens such as allergens, employing antibody or T cell receptor compositions.
Mammalian host immune response may be provoked by a variety of stimuli, including pathogens and allergens, and all involve T lymphocytes, either indirectly as helper cells which stimulate B cells to produce antibodies against these agents, or directly as T cells producing the direct response.
Poison oak and ivy dermatitis is but one example of a direct T cell mediated allergic reaction. This reaction occurs against the antigen urushiol which contacts the skin, bronchial epithelium, or the like. It s a leading cause of worker's compensation in the United States, and affects more than 80% of Americans. The antigen is an oil, and is essentially the same in poison oak, ivy, and sumac. The oil is found in the leaves and sap of the plant and is quite stable. Dermatitis can result from direct contact with the plant itself or from contact with fomites, such as the coat of a dog, or shoes worn through a patch of the plant. The allergic reaction is usually manifest as a dermatitis, but can also appear as a severe bronchitis produced by inhaling smoke from a fire containing poison oak, since the oil can coat microscopic particles in the soot and be inhaled. In some cases the reaction can be life threatening, such as with recurrent episodes which damage the skin barrier and allow systemic infections to occur. In most cases, however, it primarily affects one's ability to work (especially individuals who cannot avoid exposure as part of their work, e.g. farm workers, fire fighters, or the like) and quality of life.
As with most allergens, individuals vary in the extent of their sensitivity to urushiol. Skin testing can predict those individuals who will be most affected by the reaction. The effective amount of urushiol in one leaf is about 2 .mu.g. Those individuals developing reactions to direct application of less than 0.05 pg are termed "exquisitely sensitive". It can be predicted that they will develop dermatitis upon even minimal exposure. Those individuals requiring about 0.05-2.5 .mu.g for reaction are termed "moderately sensitive". Such individuals can tolerate modest exposure to urushiol.
It has been known for many years that oral administration of urushiol in gradually increasing doses over a period of 3-6 months can produce hyposensitization. Epstein, W., et l.,Arch. Dermatol. (1982)118:630-633. This means that the individual requires larger doses of urushiol to develop the dermatitis following hyposensitization treatment. Thus, an "exquisitely sensitive" individual can be converted to a "moderately sensitive" individual with the accompanying clinical advantages. It is necessary to monitor these patients closely, since too rapid acceleration of the doses can result in pruritus or other manifestations of allergic reaction. Although the procedure is thus rather unwieldy due to potential allergic reaction, hyposensitization in this manner can last up to one year or more. In addition, it has been shown that a single series of three weekly intramuscular injections in nonsensitized individuals can protect them from sensitization to urushiol for up to 7 years. Epstein, W., et al., J. Allergy Clin. Immunol. (1981)68:20-25. This approach is not feasible for adults since most of those who are susceptible to sensitization have already become so and intramuscular injection of urushiol in such a sensitized individual is associated with significant morbidity. Nevertheless, it does indicate that administration of antigen is capable both of blocking sensitization (tolerance) or decreasing reactivity (hyposensitization).
The active ingredient in poison oak/ivy, urushiol, is a mixture of four closely related alkylcatechols. All have the same catechol ring, and an alkyl side chain of 15 or 17 carbons, with various degrees of unsaturation ranging from zero to three double bonds. The catechol ring is capable of spontaneously forming covalent bonds with larger molecules which mount a nucleophilic attack upon the catechol ring. This is presumed to be a necessary step in activation since closely related molecules with ring structures incapable of such bond formation are not immunogenic. Urushiol is intensely hydrophobic, dissolves rapidly into the skin, and presumably penetrates rapidly into the epidermis to the Langerhans cells.
The animal models that have been most utilized for investigation of the mechanism of action of urushiol hypersensitivity dermatitis have been the guinea pig and the mouse. The guinea pig develops a direct dermatitis after sensitization and challenge. This dermatitis closely resembles the dermatitis noted in humans including erythema, induration, and blistering at higher doses. The mouse s usually sensitized by abdominal painting and challenged on the ear; reactivity is manifest as ear swelling. The guinea pig has been primarily utilized for experiments with urushiol analogues, and it was in this species that the importance of the catechol ring was demonstrated. Baer, H., et al., J. Immunol. (1970)104:178-184. The mouse, because of its inbred availability has been utilized for cell and serum transfer experiments. It was demonstrated in the mouse that urushiol reactivity could be transferred through T lymphocytes. Dunn, I., et al., Cell. Immunol. (1982):68:377-388. Following sensitization, there is a period of time (about 1 month) where challenge with urushiol or its components can still elicit a response. After this time, however, the mouse becomes refractory to challenge. Suspecting that this refractory phase represented active tolerance, it was demonstrated that T lymphocytes taken from animals during this period could transfer resistance to sensitization (tolerance) to virgin animals. Further, it was shown that serum from animals during this time were also capable of suppressing reactivity, suggesting that serum factors were present in the donor animals which either directly blocked sensitization, or produced suppressor cells which blocked sensitization, or both. Dunn, I., et al., J. Invest. Dermatol. (1987)89:296-298.
Because of its low molecular weight (320) Da urushiol falls into the class of compounds termed haptens. These chemicals can be potent immunogens (sensitizers) and are capable of inducing both T cell mediated immunity, and B cell mediated immunity (antibodies). Before they can exert this action, however, they must first be coupled to higher molecular weight molecules. Such molecules can range from serum proteins such as human serum albumin (HSA) to self cell bound molecules such as those of the major histocompatibility loci (MHC molecules). In most cases the bond must be covalent although some haptens such as nickel may be able to produce this effect by very firm non-covalent binding. Urushiol and its components are presumed to form this covalent bond spontaneously in vivo by oxidation of the catechol ring to a quinone and resultant hydrophilic attack by a larger molecule which can serve as the carrier. Since these carrier molecules can form part of the eventual antigen that is recognized by either T cells or antibody, it is often difficult to predict what the actual specificity of the immune response will be. In the case of animal models of some autoimmune diseases, such as experimental autoimmune encephalomyelitis used as a model for multiple sclerosis, the antigen causing the disease has been identified as myelin basic protein, and it is recognized in conjunction with the individual MHC molecules of each animal. Acha-Orbea, H., et al., Ann. Rev. Immunol. (1989)7:371-405. Thus the specificity of the T cell receptor (TCR) or of antibodies recognizing the complex will be different between animals with different MHC molecules. This is the case with most other animal models of autoimmune diseases. In contrast, a common MHC association site has been identified for some haptens, so it is possible that the specificity of the TCR or antibodies may be common between different animals. Baskar, S., et al., Mol. Immunol. 1990)27:79-86.
A wide variety of experimental haptens are known to produce delayed type hypersensitivity reactions (DTH). As in the case of urushiol, the tolerance can be transferred by T cells which are termed suppressor cells). Other work has demonstrated that anti-idiotypic antibodies can also transfer tolerance. Claman, H., et al., Immunol. Rev. (1980)50:105-132. It has therefore been suggested that at least one mechanism of tolerance in these cases is the generation of either anti-idiotypic antibodies, suppressor cells, or both, all of which can induce tolerance. In an animal system this tolerant state may be the usual sequelae of an immune response after antigen challenge, but appropriate manipulation of the system could allow the induction of this state without the initial dermatitis.
Antibodies reacting with a specific antigen can themselves suppress antibody responses to that antigen. This has been demonstrated with particulate antigens such as sheep erythrocytes, and proteins where administration of antibody both before or following antigen stimulation suppressed antibody responses in the recipient. Krieger-Eddy, N., et al., J. Immunol. (1987)138:1693-1698. The specificity of antibody induced immune suppression is quite variable since in some cases antibodies reacting with one epitope will suppress antibody responses to the whole antigen. In other cases antibody treatment only suppresses responses to the epitope recognized by the treating antibody. This approach for controlling immune responses to haptens is particularly attractive, since molecules such as urushiol only have a few (often no more than one) epitopes. (An epitope is the simplest form of an antigenic determinant present on a complex antigenic molecule.)
The mechanism of action of antibody mediated suppression of immune responses is not clear. Removal of antigen from the system is possible although more specific mechanisms have been proposed to account for studies where antibody treatment was effective when given after antigen stimulation. These include antibody inhibition of antigen processing at the level of interaction of antigen processing cells and interactions with T helper cells possibly by antibody binding to the T cell receptor (TCR), as well as inhibition of antigen specific B cells.
The immune system is now considered to be composed of network (idiotypic network) in which components can stimulate or down regulate immune responses. See e.g. Wigzell & Bing, Progress in Immunology IV, eds. Fougereau & Dausset (Academic Press, N.Y.) p. 94-103 (1980); Infante, et al., J. Exp. (1982)155:1100; Bona & Paul, Regulatory T Lymphocytes, eds. Pernis & Vogel (Academic Press, N.Y.) p. 292 (1980); WO 84/02848. The antigen is considered to induce an antibody (Ab.sub.1) or an immune T lymphocyte having a TCR. These in turn can stimulate anti-idiotypic antibodies (Ab.sub.2). These anti-idiotypic antibody responses generate families of antibodies recognizing the combining site of the first antibody (Ab.sub.1) and/or related structures. These anti-idiotypic antibodies provide a means for altering T cell and B cell responses, one component being down regulation of immune responses to the original antigen. Anti-idiotypic antibodies can also stimulate antibody responses to produce anti-anti-idiotypic antibodies (Ab.sub.3) which in some cases react with the original antigen.
Although anti-idiotypic antibodies have been shown multiple systems to down regulate the immune response, recent data indicates more potent reagents for this may be either the T cell receptor itself, (with specificity similar to Ab.sub.1) used as active vaccination, or anti-idiotypic antibodies against the TCR (equivalent to Ab.sub.2, used for passive therapy). The reason for this relates to the specificity of the antibody. Since antigens are recognized in association with class II MHC antigens, the actual TCR has a specificity recognizing the antigen in association with the class II MHC. In other systems this specificity is so exquisite that the specificity of a TCR against a given antigen will differ between individuals and species of inbred animals, since the portion of the MHC co-recognized is variable between individuals. However it has recently been shown that many antigens are recognized in conjunction with a region of the MHC which is common to many species (called an agretope). This has suggested that a superior immunogen may be constructed by using antigen coupled to this common portion of the MHC, which would be an effective vaccine in all individuals, and indeed in animal systems as well. In addition, an antibody made against the anti-urushiol TCR isolated from one individual should serve as an effective agent for passive immunotherapy in other individuals as well.
This hypothesis has been recently tested by demonstrating that the immunoglobulin fraction of patients hyposensitized (by oral administration) to urushiol contained a fraction which could abrogate the sensitization of mice to urushiol. This suggests that the effect was produced either by Ab.sub.1 or Ab.sub.2, which can cross species (Stampf, J.L. et. al.J. Invest. Derm. 95:363-365, 1990). If the Ab.sub.2 is directed against the TCR, as the literature would suggest is the case, this finding indicates that the reactivity of such antibodies is not even limited to the same species.
There is compelling evidence that T cells recognize antigen in the context of MHC molecule, this occurs on the surface of antigen presenting cells. A strong correlation between MHC restrict-on of response to a particular antigenic determinant and its ability to bind to purified MHC molecules has been demonstrated. Buus, S., et al., Immunol. Rev. (1987)98:115-141. The small synthetic hapten L-tyrosine p-azobenzenearsonate (ABA-tyr) is immunogenic n several mouse haplotypes and it has been shown that the azo linked benzene groups of the hapten function as an agretope. Based upon a hypothetical model of the MHC class II protein (Brown, J. H., et al., Nature (1988)329:845) the hydrophobic residues (Tyr.sup.30, Ile.sup.31, Tyr.sup.32, Tyr.sup.37, and Val.sup.38) in the B.sub.1 domain form a hydrophobic patch on the binding cleft and have been suggested as likely contact points for the agretope of ABA-tyr. Goodman, J. W., Chem. Immunol. (1989)46:1. This hydrophobic region is a conserved characteristic of all Ia molecules.
The allergenic properties of alkylcatechols such as urushiol are dependent upon their reactivity towards nucleophiles such as amino or thiol groups of amino acids. Baer, et al., J. Immunol (1967)99:365. In addition, allergic potency is dependent upon the side chain and the presence of unsaturated bonds. From these findings it has been predicted that the hydrophobic allergen urushiol interacts with a common internal agretope on MHC molecules by covalent bond formation through the quinone ring and hydrophobic structures between the alkyl side chain and MHC with MHC restriction being minimal. This has led to several approaches for inducing immune responses to these haptens. These include formation of urushiol conjugates with MHC protein preparations as well as peptides containing sequences like the predicted internal agretope. Other approaches involve treating antigen processing cells such as cultured human Langerhans cells (Romani, N., et al., J. Invest. Derm. (1989)93:600-09) or mouse peritoneal macrophages with urushiol.
The present invention relates to the down regulation of immune response to exogenous antigens such as the allergen urushiol by manipulating the idiotypic network. This can be effected by the use of TCR's, Ab.sub.1, or Ab.sub.2, all with specificity directed against the antigen either alone or in combination with other carriers. The advantage of this more direct approach over older methods is that side effects related to prolonged antigen administration are avoided, the procedure is faster and more effective, and the use of limited epitopes make the procedure feasible in cases where antigen hyposensitization is not possible. Urushiol is used as an example of the procedures, which should be applicable to any exogenous antigen or allergen in which dominant epitope(s) can be identified.